Sample processing units, systems, and related methods

ABSTRACT

Sample processing units useful for mixing and purifying materials, such as fluidic materials are provided. A sample processing unit typically includes a container configured to contain a sample comprising magnetically responsive particles, and one or more magnets that are in substantially fixed positions relative to the container. A sample processing unit also generally includes a conveyance mechanism configured to convey the container to and from a position that is within magnetic communication with the magnet, e.g., such that magnetically responsive particles with captured analytes can be retained within the container when other materials are added to and/or removed from the container. Further, a sample processing unit also typically includes a rotational mechanism that is configured to rotate the container, e.g., to effect mixing of sample materials disposed within the container. Related carrier mechanisms, sample processing stations, systems, and methods are also provided.

The present application is a continuation of U.S. patent applicationSer. No. 13/595,189 filed Aug. 27, 2012, now U.S. Pat. No. 8,609,430,issued Dec. 17, 2013, which is a continuation of U.S. patent applicationSer. No. 13/221,608 filed Aug. 30, 2011, now U.S. Pat. No. 8,252,599,issued Aug. 28, 2012, which is a continuation of U.S. patent applicationSer. No. 12/561,175 filed Sep. 16, 2009, now U.S. Pat. No. 8,148,163,issued Apr. 3, 2012, which claims priority to U.S. ProvisionalApplication Ser. No. 61/097,525 filed Sep. 16, 2008, each of which isherein incorporated by reference in its entirety.

FIELD OF THE INVENTION

The invention relates generally to sample purification, and providessample processing units, systems, and methods useful for this purpose.

BACKGROUND OF THE INVENTION

Nucleic acid amplification techniques, such as the polymerase chainreaction (PCR) have widespread applications in many scientificdisciplines, including microbiology, medical research, forensicanalysis, and clinical diagnostics. In some of these applications, PCRproducts are “sized” using traditional biochemical techniques such asstandard gel electrophoresis involving either intercalating dyes orfluorescently labeled primers. Other applications, such as 5′-nucleaseor TaqMan® probe-based assays, which are widely used in a number ofPCR-related diagnostic kits, confirm the presence (or absence) of aspecific PCR product, but provide no direct information on the size ofthe particular amplicon. These methods typically have limited utilityfor relatively small amplicons (less than 150 base pairs), owing to theproportionately high fluorescence background, and do not provide anyinformation with respect to amplicon heterogeneity or exact length.

Electrospray ionization mass spectrometry (ESI-MS) has become animportant technique for the analysis of biopolymers, including nucleicacids. Compared to the more traditional nucleic acid analysis methodsmentioned above, ESI-MS as a platform on which to characterize PCRproducts typically provides improved speed, sensitivity, and massaccuracy, among other attributes. Further, since the exact mass of eachnucleotide or nucleobase is known with great accuracy, a high-precisionmass measurement obtained via mass spectrometry can be used to derive abase composition within the experimentally obtained mass measurementuncertainty. In certain applications, the base compositions of PCRproducts are used to identify unknown bioagents, genotype nucleic acids,and provide drug resistance profiles as well as other information aboutthe corresponding template nucleic acids or source organisms.

In the electrospray ionization process, large charged droplets areproduced in the process of “pneumatic nebulization” where the analytesolution is forced through a needle at the end of which is applied apotential sufficient to disperse the emerging solution into a very finespray of charged droplets all of which have the same polarity. Thesolvent evaporates, shrinking the droplet size and increasing the chargeconcentration at the droplet's surface. Eventually, at the Rayleighlimit, Coulombic repulsion overcomes the droplet's surface tension andthe droplet explodes. This “Coulombic explosion” forms a series ofsmaller, lower charged droplets. The process of shrinking followed byexplosion is repeated until individually charged analyte ions areformed. The charges are statistically distributed amongst the analyte'savailable charge sites, leading to the possible formation of multiplycharged ions. Increasing the rate of solvent evaporation, by introducinga drying gas flow counter current to the sprayed ions, increases theextent of multiple-charging. Decreasing the capillary diameter andlowering the analyte solution flow rate, e.g., in nanospray ionization,typically creates ions with higher mass-to-charge (m/z) ratios (i.e., itis a softer ionization technique) than those produced by “conventional”ESI and are commonly used in the field of bioanalysis.

ESI generally requires relatively clean samples and is intolerable ofcationic salts, detergents, and many buffering agents commonly used inbiochemical laboratories. Buffer systems commonly employed in polymerasechain reactions, for example, typically include electrosprayincompatible reagents such as KCl, MgCl₂, Tris-HCl, and each of the fourdeoxynucleotide triphosphates (dNTPs). Even the presence of relativelylow concentrations of metal cations (e.g., less than 100 μM) can reduceMS sensitivity for oligonucleotides as the signal for each molecular ionis spread out over multiple salt adducts. Thus, in addition to removingdetergents and dNTPs, effective ESI-MS of PCR products typicallyrequires that the salt concentration be reduced by more than a factor of1000 prior to analysis.

Ethanol precipitation has been used to desalt PCR products forsubsequent MS analysis as short oligonucleotides and salts are removedwhile the sample is concentrated. In some of these methods, the PCRproduct can be precipitated from concentrated ammonium acetatesolutions, either overnight at 5° C. or over the course of 10-15 minuteswith cold (−20° C.) ethanol. Unfortunately, a precipitation step aloneis generally insufficient to obtain PCR products which are adequatelydesalted to obtain high-quality ESI spectra; consequently, precipitationis generally followed by a dialysis step to further desalt the sample.While several approaches have successfully employed these methods tocharacterize a number of PCR products, there remains a need to applythese and related methods in a robust and fully automatedhigh-throughput manner.

SUMMARY OF THE INVENTION

The present invention provides sample processing units that are usefulin various purification processes. In certain embodiments, for example,the sample processing units are used to perform solution capture methodsof purifying nucleic acids, which are subsequently analyzed using anysuitable approach, including electrospray mass spectrometric-basedanalysis. Some of these embodiments include adding an anion exchangeresin to the solution and mixing these materials in a sample processingunit to yield a suspension of the anion exchange resin in the solutionin which the nucleic acids bind to the anion exchange resin. Additionalprocessing steps performed using the sample processing units describedherein typically include isolating the anion exchange resin from thesolution, washing the anion exchange resin to remove one or morecontaminants with one or more wash buffers while retaining the nucleicacids bound to the resin, and eluting the nucleic acids from the anionexchange resin with an elution buffer, thereby yielding purified nucleicacids that are suitable for further analysis. In addition to sampleprocessing units and stations, the invention also provides relatedsystems and methods.

In one aspect, the invention provides a sample processing unit thatincludes at least one container (e.g., a cuvette or the like) configuredto contain at least one sample comprising at least one magneticallyresponsive particle (e.g., a magnetically responsive bead coated with aselected capture reagent, etc.), and at least one magnet (e.g., apermanent magnet, an electromagnet, etc.) that generates, or isconfigured to generate, at least one magnetic field, which magnet is ina substantially fixed position relative to the container. The sampleprocessing unit also includes at least one conveyance mechanismconfigured to convey the container between at least first and secondpositions in which at least the first position is within magneticcommunication with the magnet when the magnet generates the magneticfield, and at least one rotational mechanism operably connected to thecontainer, which rotational mechanism is configured to rotate thecontainer when the container is in at least the second position.Typically, the sample processing unit includes at least one mountingbracket that is operably connected to one or more of: the container, themagnet, the conveyance mechanism, or the rotational mechanism. In someembodiments, a sample processing station includes the sample processingunit. In certain embodiments, a carrier mechanism includes the sampleprocessing unit. In some of these embodiments, a system includes thecarrier mechanism.

In some embodiments, the conveyance mechanism comprises at least onemotor. The conveyance mechanism is configured to rotate the containerbetween the first and second positions in certain embodiments. In otherexemplary embodiments, the conveyance mechanism is configured to slidethe container between the first and second positions. To furtherillustrate, in some embodiments, the conveyance mechanism comprises atleast one support member operably connected to the container and/or tothe rotational mechanism. In some of these embodiments, the supportmember is configured to slide between the first and second positions,whereas in other exemplary embodiments, the support member is configuredto rotate between the first and second positions.

In certain embodiments, the rotational mechanism is configured to rotatethe container in at least one pulsed mode, during which a substantialportion of the time of rotation, a rate of rotation of the containerexceeds a rate of rotation of the sample when the container contains thesample such that the sample is sheared away from a surface of thecontainer. In some embodiments, the rotational mechanism is configuredto rotate the container in at least one oscillating motion.

In another aspect, the invention provides a sample processing unit thatincludes at least one cuvette configured to contain at least one samplecomprising at least one magnetically responsive particle, and at leastone magnet (e.g., a permanent magnet, an electromagnet, etc.) thatgenerates, or is configured to generate, at least one magnetic field,which magnet is in a substantially fixed position. In some embodiments,two or more magnets are disposed proximal to a receiving space in whichthe cuvette is located at least partially within the receiving spacewhen the cuvette is in the first position. The sample processing unitalso includes at least a first motor operably connected to the cuvette.The first motor is configured to rotate the cuvette around a centrallongitudinal axis of the cuvette. In addition, the sample processingunit also includes at least one support member (e.g., a swing arm or thelike) operably connected to the first motor, and at least a second motoroperably connected to the support member. The second motor is configuredto rotate the cuvette between at least first and second positions inwhich at least the first position is within magnetic communication withthe magnet when the magnet generates the magnetic field. In certainembodiments, a sample processing station includes the sample processingunit. Optionally, a carrier mechanism (e.g., a carousel, a conveyortrack, etc.) includes the sample processing unit. In some of theseembodiments, a system includes the carrier mechanism.

In some embodiments, the first motor (e.g., a stepper motor, a servomotor, etc.) is configured to rotate the cuvette in at least one pulsedmode, during which a substantial portion of the time of rotation, a rateof rotation of the cuvette exceeds a rate of rotation of the sample whenthe cuvette contains the sample such that the sample is sheared awayfrom a surface of the cuvette, e.g., to effect thorough mixing of thesample and other materials that may be present in the cuvette. Tofurther illustrate, the first motor is optionally configured to rotatethe cuvette in at least one oscillating motion. In certain embodiments,the second motor comprises a brushless direct current motor or the like.The sample processing unit generally includes circuitry configured tocontrol the first and second motors.

In certain embodiments, the support member comprises a first end and asecond end in which the cuvette is retained at or proximal to the firstend of the support member, and in which the second motor is operablyconnected to the support member at or proximal to the second end of thesupport member. In some of these embodiments, the support member isconfigured to rotate at least partially around a rotational axisextending through the second end of the support member. As a furtherillustrate, in some embodiments, a pin is fixedly coupled to the secondend of the support member and aligned with the rotational axis in whichthe pin is operably coupled to the second motor.

In some embodiments, the sample processing unit includes a mountingbracket in which the support member is operably connected to themounting bracket. In certain of these embodiments, the magnet isoperably connected to the mounting bracket.

In another aspect, the invention provides a sample processing system.The system includes at least one sample processing unit that comprises:at least one cuvette configured to contain at least one samplecomprising at least one magnetically responsive particle; at least onemagnet that generates, or is configured to generate, at least onemagnetic field, which magnet is in a substantially fixed position; atleast a first motor operably connected to the cuvette, which first motoris configured to rotate the cuvette around a central longitudinal axisof the cuvette; at least one support member operably connected to thefirst motor; and at least a second motor operably connected to thesupport member, which second motor is configured to rotate the cuvettebetween at least first and second positions in which at least the firstposition is within magnetic communication with the magnet when themagnet generates the magnetic field. The system also includes at leastone carrier mechanism operably connected to the sample processing unit.The carrier mechanism is configured to move the sample processing unitto one or more locations. The system further includes at least onematerial transfer component configured to transfer material to and/orfrom the cuvette, and at least one controller operably connected to thesample processing unit, the carrier mechanism, and/or the materialtransfer component. The controller is configured to effect one or moreof: the magnet to generate the magnetic field, the first motor to rotatethe cuvette, the second motor to rotate the cuvette between the firstand second positions, the carrier mechanism to move the sampleprocessing unit to the one or more locations, or the material transfercomponent to transfer the material to and/or from the cuvette.

The carrier mechanism includes various embodiments. In one embodiment,for example, the carrier mechanism comprises a carousel that isconfigured to rotate the sample processing unit to the one or morelocations. In another exemplary embodiment, the carrier mechanismcomprises a conveyor track that is configured to convey the sampleprocessing unit to the one or more locations. Typically, the carriermechanism comprises a plurality of sample processing units. In some ofthese embodiments, the material transfer component comprises a manifoldthat is configured to transfer material to and/or from the cuvettes ofat least two sample processing units substantially simultaneously.

In some embodiments, the material transfer component comprises one ormore of: a sample input gantry head, a sample wash station, a sampleoutput gantry head, or a cuvette wash station. Typically, the materialtransfer component is configured to transfer fluidic material. Incertain embodiments, the material transfer component comprises one ormore needles.

In certain embodiments, the controller is configured to effect the firstmotor to rotate the cuvette in one or more selectable modes. In someembodiments, for example, the controller is configured to effect thefirst motor to rotate the cuvette in at least one pulsed mode, duringwhich a substantial portion of the time of rotation, a rate of rotationof the cuvette exceeds a rate of rotation of the sample when the cuvettecontains the sample such that the sample is sheared away from a surfaceof the cuvette. In other exemplary embodiment, the controller isconfigured to effect the first motor to rotate the cuvette in at leastone oscillating motion.

In some embodiments, the sample processing system includes at least onedetector configured to detect one or more detectable signals of or fromone or more sample components. In certain embodiments, the detector iswithin sensory communication with the cuvette when the carrier mechanismmoves the sample processing unit to at least one of the locations.Optionally, the material transfer component is configured to transferthe material from the cuvette to the detector. In some embodiments, thecontroller is operably connected to the detector. In these embodiments,the controller is configured to effect the detector to detect thedetectable signals of or from the sample components. To furtherillustrate, in certain embodiments, the detector comprises a massspectrometer. In some of these embodiments, the mass spectrometercomprises an electrospray ionization time-of-flight mass spectrometer.

In another aspect, the invention relates to a method of processing asample. The method includes (a) providing at least one sample processingunit that comprises: at least one cuvette that contains at least onesample comprising at least one magnetically responsive particlecomprising at least one captured first component (e.g., a biopolymer,such as a polynucleotide, a polypeptide, or the like); at least onemagnet that is in a substantially fixed position; at least a first motoroperably connected to the cuvette, which first motor is configured torotate the cuvette around a central longitudinal axis of the cuvette; atleast one support member operably connected to the first motor; and atleast a second motor operably connected to the support member, whichsecond motor is configured to rotate the cuvette between at least firstand second positions in which the magnet is capable of magneticallycommunicating with the cuvette when the cuvette is at least in the firstposition. The method also includes (b) moving the cuvette into the firstposition using the second motor such that a magnetic field generated bythe magnet causes the magnetically responsive particle to move and/or beretained proximal to a surface of the cuvette. In addition, the methodalso includes (c) removing at least a second component from the cuvetteto thereby process the sample. In some embodiments, the method includesadding the sample and/or the magnetically responsive particle to thecuvette prior to (a) when the cuvette is in the second position.Optionally, the method includes adding at least one wash reagent to thecuvette. In certain embodiments, the magnet comprises a permanentmagnet. In other exemplary embodiments, the magnet comprises anelectromagnet. In these embodiments, the method typically comprisesgenerating the magnetic field prior to or during (b). Typically, acarrier mechanism comprises the sample processing unit and the methodcomprises moving the sample processing unit to one or more locations.

The magnetically responsive particle includes various embodiments. Insome embodiments, for example, the magnetically responsive particlecomprises an anion exchange resin. Typically, the magneticallyresponsive particle comprises at least one biopolymer capture reagent.In certain embodiments, the biopolymer capture reagent comprises atleast one anion exchange functional group. The anion exchange functionalgroup typically comprises a pKa value of 9.0 or greater. To furtherillustrate, exemplary anion exchange functional groups are selectedfrom, e.g., a primary amine, a second amine, a tertiary amine, aquaternary amine, a polyethyleneimine, a charged aromatic amine, adiethylaminomethyl, a diethylaminoethyl.

The method typically includes rotating the cuvette using the first motorsuch that sample components mix with one another. In some embodiments,the method includes rotating the cuvette when the cuvette is in thesecond position. In certain embodiments, the method includes rotatingthe cuvette in at least one pulsed mode, during which a substantialportion of the time of rotation, a rate of rotation of the cuvetteexceeds a rate of rotation of the sample such that the sample is shearedaway from a surface of the cuvette. Optionally, the method includesrotating the cuvette in at least one oscillating motion.

In certain embodiments, the method includes detecting at least onedetectable signal of or from the sample. For example, the methodoptionally includes detecting a molecular mass of the first component.In these embodiments, the molecular mass is generally detected using amass spectrometer. In some of these embodiments, the first componentcomprises a nucleic acid and the method comprises correlating themolecular mass of the nucleic acid with a base composition and/or anidentity of the nucleic acid.

BRIEF DESCRIPTION OF THE DRAWINGS

The description provided herein is better understood when read inconjunction with the accompanying drawings which are included by way ofexample and not by way of limitation. It will be understood that likereference numerals identify like components throughout the drawings,unless the context indicates otherwise. It will also be understood thatsome or all of the figures may be schematic representations for purposesof illustration and do not necessarily depict the actual relative sizesor locations of the elements shown.

FIG. 1A schematically shows a sample processing unit with a cuvette in asecond position from a perspective view according to one embodiment ofthe invention.

FIG. 1B schematically depicts the sample processing unit of FIG. 1A withthe cuvette in a first position from a perspective view.

FIG. 1C schematically depicts the sample processing unit of FIG. 1A withthe cuvette in a first position from a side elevation view.

FIG. 1D schematically illustrates a detailed side elevation view of amotor operably connected to a cuvette of the sample processing unit ofFIG. 1A.

FIG. 2 is a mass spectrum obtained for a 65-mer PCR product following apurification and desalting protocol described herein. The two peakscorrespond to sense and antisense strands of the PCR amplicons, whichseparate under the conditions of ESI. Low amplitude salt adductsindicated effective cleanup of the PCR product.

FIG. 3A schematically shows a sample processing unit with a slidablesupport member in a first position from a front elevation view accordingto one embodiment of the invention.

FIG. 3B schematically depicts the sample processing unit of FIG. withthe slidable support member in a second position from a perspectiveview.

FIG. 4A schematically illustrates a carrier mechanism with a manifoldfrom a perspective view according to one embodiment of the invention.

FIG. 4B schematically shows the carrier mechanism and manifold of FIG.4A from a side elevation view.

FIG. 4C schematically shows the carrier mechanism and manifold of FIG.4A from a top view.

FIG. 4D schematically illustrates a detailed perspective view of thecarrier mechanism and manifold of FIG. 4A.

FIG. 4E schematically depicts a detailed side elevation view of thecarrier mechanism and manifold of FIG. 4A.

FIG. 4F schematically depicts a detailed front elevation view of thecarrier mechanism and manifold of FIG. 4A.

FIG. 5A schematically illustrates a carrier mechanism that includes aconveyor track from a top view according to one embodiment of theinvention.

FIG. 5B schematically illustrates the carrier mechanism from FIG. 5Afrom a side elevation view.

FIG. 6 is a block diagram showing a representative logic device in whichvarious aspects of the present invention may be embodied.

FIG. 7A schematically illustrates selected components of arepresentative system that includes a sample processing station as asub-system component from a perspective view according to one embodimentof the invention.

FIG. 7B schematically shows the representative system of FIG. 7A from afront elevation view.

FIG. 7C schematically depicts the representative system of FIG. 7A froma rear elevation view.

FIG. 7D schematically shows the representative system of FIG. 7A from aside elevation view.

FIG. 7E schematically illustrates the representative system of FIG. 7Afrom a top elevation view.

FIG. 7F schematically depicts the representative system of FIG. 7A froma cross-sectional view.

FIG. 7G schematically illustrates the representative system of FIG. 7Afrom a cross-sectional view.

FIG. 8A and FIG. 8B schematically show additional components of arepresentative system of FIG. 7A from a perspective view.

FIG. 9A schematically illustrates the representative system of FIG. 7Awith an external covering from a perspective view.

FIG. 9B schematically illustrates the representative system of FIG. 7Awith an external covering from a front elevation view.

FIG. 9C schematically shows the representative system of FIG. 7A with anexternal covering from a side view.

DETAILED DESCRIPTION A. Definitions

Before describing the invention in detail, it is to be understood thatthis invention is not limited to particular sample processing units,systems, or methods, which can vary. As used in this specification andthe appended claims, the singular forms “a,” “an,” and “the” alsoinclude plural referents unless the context clearly provides otherwise.Thus, for example, reference to “a sample processing unit” includes acombination of two or more sample processing units. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting. Further, unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionpertains. In describing and claiming the invention, the followingterminology, and grammatical variants thereof, will be used inaccordance with the definitions set forth below.

The term “amplifying” or “amplification” in the context of nucleic acidsrefers to the production of multiple copies of a polynucleotide, or aportion of the polynucleotide, typically starting from a small amount ofthe polynucleotide (e.g., a single polynucleotide molecule), where theamplification products or amplicons are generally detectable.Amplification of polynucleotides encompasses a variety of chemical andenzymatic processes. The generation of multiple DNA copies from one or afew copies of a target or template DNA molecule during a polymerasechain reaction (PCR) or a ligase chain reaction (LCR) are forms ofamplification. Amplification is not limited to the strict duplication ofthe starting molecule. For example, the generation of multiple cDNAmolecules from a limited amount of RNA in a sample using reversetranscription (RT)-PCR is a form of amplification. Furthermore, thegeneration of multiple RNA molecules from a single DNA molecule duringthe process of transcription is also a form of amplification.

The term “base composition” refers to the number of each residuecomprised in an amplicon or other nucleic acid, without considerationfor the linear arrangement of these residues in the strand(s) of theamplicon. The amplicon residues comprise, adenosine (A), guanosine (G),cytidine, (C), (deoxy)thymidine (T), uracil (U), inosine (I),nitroindoles such as 5-nitroindole or 3-nitropyrrole, dP or dK (Hill Fet al. (1998) “Polymerase recognition of syntheticoligodeoxyribonucleotides incorporating degenerate pyrimidine and purinebases” Proc Natl Acad Sci U.S.A. 95(8):4258-63), an acyclic nucleosideanalog containing 5-nitroindazole (Van Aerschot et al., Nucleosides andNucleotides, 1995, 14, 1053-1056), the purine analog1-(2-deoxy-beta-D-ribofuranosyl)-imidazole-4-carboxamide,2,6-diaminopurine, 5-propynyluracil, 5-propynylcytosine, phenoxazines,including G-clamp, 5-propynyl deoxy-cytidine, deoxy-thymidinenucleotides, 5-propynylcytidine, 5-propynyluridine and mass tag modifiedversions thereof, including 7-deaza-2′-deoxyadenosine-5-triphosphate,5-iodo-2′-deoxyuridine-5′-triphosphate,5-bromo-2′-deoxyuridine-5′-triphosphate,5-bromo-2′-deoxycytidine-5′-triphosphate,5-iodo-2′-deoxycytidine-5′-triphosphate,5-hydroxy-2′-deoxyuridine-5′-triphosphate,4-thiothymidine-5′-triphosphate, 5-aza-2′-deoxyuridine-5′-triphosphate,5-fluoro-2′-deoxyuridine-5′-triphosphate,0⁶-methyl-2′-deoxyguanosine-5′-triphosphate,N²-methyl-2′-deoxyguanosine-5′-triphosphate,8-oxo-2′-deoxyguanosine-5′-triphosphate orthiothymidine-5′-triphosphate. In some embodiments, the mass-modifiednucleobase comprises ¹⁵N or ¹³C or both ¹⁵N and ¹³C. In someembodiments, the non-natural nucleosides used herein include5-propynyluracil, 5-propynylcytosine and inosine. Herein the basecomposition for an unmodified DNA amplicon is notated asA_(w)G_(x)C_(y)T_(z), wherein w, x, y and z are each independently awhole number representing the number of said nucleoside residues in anamplicon. Base compositions for amplicons comprising modifiednucleosides are similarly notated to indicate the number of said naturaland modified nucleosides in an amplicon. Base compositions arecalculated from a molecular mass measurement of an amplicon, asdescribed below. The calculated base composition for any given ampliconis then compared to a database of base compositions. A match between thecalculated base composition and a single database entry reveals theidentity of the bioagent.

The term “biopolymer” refers a biomolecule that includes at least twomonomer units attached to one another. A “biomolecule” refers to anorganic molecule that is made and/or used by an organism, and/or that isutilized to analyze the organism or components thereof. Exemplarybiomolecules include nucleic acids, nucleotides, amino acids,polypeptides, peptides, peptide fragments, sugars, fatty acids,steroids, lipids, and combinations of these biomolecules (e.g.,glycoproteins, ribonucleoproteins, or lipoproteins).

The term “captured” in the context of solid supports and target analytesrefers to an analyte that is directly or indirectly joined or attachedto a solid support via a covalent linkage, chelation, ionic interaction,and/or another mechanism. In some embodiments, target analytes arereversibly captured on solid supports, whereas in other embodiments,they are permanently captured.

The term “communicate” refers to the direct or indirect transfer ortransmission, and/or capability of directly or indirectly transferringor transmitting, something at least from one thing to another thing.Objects “fluidly communicate” with one another when fluidic material is,or is capable of being, transferred from one object to another. In someembodiments, for example, material transfer components add and/or removematerials to/from the containers of sample processing units during agiven purification process. Objects are in “magnetic communication” withone another when one object exerts or can exert a magnetic field ofsufficient strength on another object to effect a change (e.g., a changein position or other movement) in the other object. In some embodiments,for example, containers (e.g., cuvettes, etc.) of sample processingunits are moved sufficiently proximal to sample processing unit magnetssuch that magnetic fields generated by the magnets cause magneticallyresponsive particles (e.g., magnetically responsive beads with bound orotherwise captured nucleic acids) disposed within the containers to moveand/or be retained proximal to surfaces of the containers when a givenpurification processing is being performed. Objects are in “sensorycommunication” when a characteristic or property of one object is or canbe sense, perceived, or otherwise detected by another object. In certainembodiments, for example, carrier mechanisms of the systems describedherein rotate the containers of sample processing units within sensorycommunication with detectors to detect one or more parameters (e.g.,fluorescence, temperature, pH, or the like) of fluidic materialsdisposed in the containers.

The term “material” refers to something comprising or consisting ofmatter. The term “fluidic material” refers to material (such as, aliquid or a gas) that tends to flow or conform to the outline of itscontainer.

The term “microplate” refers to a plate or other support structure thatincludes multiple cavities or wells that are structured to containmaterials, such as fluidic materials. The wells typically have volumecapacities of less than about 1.5 mL (e.g., about 1000 mL, about 800 mL,about 600 mL, about 400 mL, or less), although certain microplates(e.g., deep-well plates, etc.) have larger volume capacities, such asabout 4 mL per well. Microplates can include various numbers of wells,for example, 6, 12, 24, 48, 96, 384, 1536, 3456, 9600, or more wells. Inaddition, the wells of a microplate are typically arrayed in arectangular matrix. Microplates generally conform to the standardspublished by the American National Standards Institute (ANSI) on behalfof the Society for Biomolecular Screening (SBS), namely, ANSI/SBS1-2004: Microplates—Footprint Dimensions, ANSI/SBS 2-2004:Microplates—Height Dimensions, ANSI/SBS 3-2004: Microplates—BottomOutside Flange Dimensions, and ANSI/SBS 4-2004: Microplates—WellPositions, which are each incorporated by reference. Microplates areavailable from a various manufacturers including, e.g., Greiner AmericaCorp. (Lake Mary, Fla., U.S.A.) and Nalge Nunc International (Rochester,N.Y., U.S.A.), among many others. Microplates are also commonly referredto by various synonyms, such as “microtiter plates,” “micro-wellplates,” “multi-well containers,” and the like

The term “molecular mass” refers to the mass of a compound as determinedusing mass spectrometry, for example, ESI-MS. Herein, the compound ispreferably a nucleic acid. In some embodiments, the nucleic acid is adouble stranded nucleic acid (e.g., a double stranded DNA nucleic acid).In some embodiments, the nucleic acid is an amplicon. When the nucleicacid is double stranded the molecular mass is determined for bothstrands. In one embodiment, the strands may be separated beforeintroduction into the mass spectrometer, or the strands may be separatedby the mass spectrometer (for example, electro-spray ionization willseparate the hybridized strands). The molecular mass of each strand ismeasured by the mass spectrometer.

The term “non-priority microplate” refers to a microplate that isprocessed or otherwise handled after at least one other microplate, orwhose processing or handling is interrupted or deferred in order toprocess or otherwise handle at least one other microplate, in a givenmicroplate handling system of the invention. That is, the order,schedule, or timing of processing or handling a non-priority microplateis subject to interruption or delay when a higher priority microplate ispresented, such as a microplate including stat samples. In someembodiments, non-priority microplates are introduced into a given systemvia non-priority microplate storage units.

The term “nucleic acid molecule” refers to any nucleic acid containingmolecule, including but not limited to, DNA or RNA. The term encompassessequences that include any of the known base analogs of DNA and RNAincluding, but not limited to, 4-acetylcytosine,8-hydroxy-N⁶-methyladenosine, aziridinylcytosine, pseudoisocytosine,5-(carboxyhydroxyl-methyl)-uracil, 5-fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethyl-aminomethyluracil, dihydrouracil, inosine,N⁶-isopentenyladenine, 1-methyladenine, 1-methylpseudo-uracil,1-methylguanine, 1-methylinosine, 2,2-dimethyl-guanine, 2-methyladenine,2-methylguanine, 3-methyl-cytosine, 5-methylcytosine, N⁶-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxy-amino-methyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonylmethyluracil, 5-methoxyuracil,2-methylthio-N-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and2,6-diaminopurine.

The term “priority microplate” refers to a microplate that is processedor otherwise handled before the processing or handling of a non-prioritymicroplate is commenced or completed in a given microplate handlingsystem of the invention. In some embodiments, one or more wells ofpriority microplates comprise stat or urgent samples. In certainembodiments, priority microplates are introduced into a given system viapriority microplate storage units.

The phrase “solid support” refers to a solid material that can bederivatized with, or otherwise attached to, a chemical or biochemicalmoiety. Exemplary solid supports that are optionally utilized includematrices and particles free in solution, such as glass (e.g., controlledpore glass (CPG)), nitrocellulose, polyacrylate, nylon, mixed polymers,silane polypropylene, polystyrene, magnetically attractable orresponsive particles (e.g., magnetic spheres or beads), and the like.

The term “system” refers a group of objects and/or devices that form anetwork for performing a desired objective. In some embodiments, forexample, sample processing units having fluidic materials withmagnetically responsive particles are included as part of systems inwhich nucleic acids are purified using the magnetically responsiveparticles such that the molecular masses of the nucleic acids can bemore readily detected by mass spectrometers of these systems.

II. Introduction

The invention relates to sample purification, and in various embodimentsprovides sample processing units, carrier mechanisms, sample processingstations, systems, and related methods that are useful for this purpose.The sample processing units and related aspects of the invention can beused, or adapted for use, in a wide variety of sample purificationprocesses. In certain embodiments, for example, microplates comprisingnucleic acid amplification reaction mixtures are loaded into microplatestorage units of a microplate handling system. In some of theseembodiments, a microplate transport mechanism of the system transportsthe microplates to a microplate processing area, where a materialtransfer component transfers aliquots of the reaction mixtures from thewells of the microplates to a sample processing system. In theseembodiments, the sample processing system is typically used to purifyamplification products or amplicons in the reaction mixture aliquots forsubsequent detection or other analysis. To further illustrate, in someof these embodiments, the molecular masses of these purified ampliconsare measured using a mass spectrometer, e.g., an electrospray ionizationtime-of-flight mass spectrometer or the like. The base compositions ofthe amplicons are typically determined from the measured molecularmasses and correlated with an identity or source of target nucleic acidsin the amplification reaction mixtures, such as a pathogenic organism.

Particular embodiments of molecular mass-based detection methods andother aspects that are optionally adapted for use with the sampleprocessing units and related aspects of the invention are described invarious patents and patent applications, including, for example, U.S.Pat. Nos. 7,108,974; 7,217,510; 7,226,739; 7,255,992; 7,312,036; and7,339,051; and US patent publication numbers 2003/0027135; 2003/0167133;2003/0167134; 2003/0175695; 2003/0175696; 2003/0175697; 2003/0187588;2003/0187593; 2003/0190605; 2003/0225529; 2003/0228571; 2004/0110169;2004/0117129; 2004/0121309; 2004/0121310; 2004/0121311; 2004/0121312;2004/0121313; 2004/0121314; 2004/0121315; 2004/0121329; 2004/0121335;2004/0121340; 2004/0122598; 2004/0122857; 2004/0161770; 2004/0185438;2004/0202997; 2004/0209260; 2004/0219517; 2004/0253583; 2004/0253619;2005/0027459; 2005/0123952; 2005/0130196 2005/0142581; 2005/0164215;2005/0266397; 2005/0270191; 2006/0014154; 2006/0121520; 2006/0205040;2006/0240412; 2006/0259249; 2006/0275749; 2006/0275788; 2007/0087336;2007/0087337; 2007/0087338 2007/0087339; 2007/0087340; 2007/0087341;2007/0184434; 2007/0218467; 2007/0218467; 2007/0218489; 2007/0224614;2007/0238116; 2007/0243544; 2007/0248969; WO2002/070664; WO2003/001976;WO2003/100035; WO2004/009849; WO2004/052175; WO2004/053076;WO2004/053141; WO2004/053164; WO2004/060278; WO2004/093644; WO2004/101809; WO2004/111187; WO2005/023083; WO2005/023986; WO2005/024046;WO2005/033271; WO2005/036369; WO2005/086634; WO2005/089128;WO2005/091971; WO2005/092059; WO2005/094421; WO2005/098047;WO2005/116263; WO2005/117270; WO2006/019784; WO2006/034294;WO2006/071241; WO2006/094238; WO2006/116127; WO2006/135400;WO2007/014045; WO2007/047778; WO2007/086904; and WO2007/100397;WO2007/118222, which are each incorporated by reference as if fully setforth herein.

Exemplary molecular mass-based analytical methods and other aspects ofuse in the sample processing units and systems described herein are alsodescribed in, e.g., Ecker et al. (2005) “The Microbial Rosetta StoneDatabase: A compilation of global and emerging infectious microorganismsand bioterrorist threat agents” BMC Microbiology 5(1):19; Ecker et al.(2006) “The Ibis T5000 Universal Biosensor: An Automated Platform forPathogen Identification and Strain Typing” JALA 6(10:341-351; Ecker etal. (2006) “Identification of Acinetobacter species and genotyping ofAcinetobacter baumannii by multilocus PCR and mass spectrometry” J ClinMicrobiol. 44(8):2921-32; Ecker et al. (2005) “Rapid identification andstrain-typing of respiratory pathogens for epidemic surveillance” ProcNatl Acad Sci USA. 102(22):8012-7; Hannis et al. (2008) “High-resolutiongenotyping of Campylobacter species by use of PCR and high-throughputmass spectrometry” J Clin Microbiol. 46(4):1220-5; Blyn et al. (2008)“Rapid detection and molecular serotyping of adenovirus by use of PCRfollowed by electrospray ionization mass spectrometry” J Clin Microbiol.46(2):644-51; Sampath et al. (2007) “Global surveillance of emergingInfluenza virus genotypes by mass spectrometry” PLoS ONE 2(5):e489;Sampath et al. (2007) “Rapid identification of emerging infectiousagents using PCR and electrospray ionization mass spectrometry” Ann N YAcad. Sci. 1102:109-20; Hall et al. (2005) “Base composition analysis ofhuman mitochondrial DNA using electrospray ionization mass spectrometry:a novel tool for the identification and differentiation of humans” AnalBiochem. 344(1):53-69; Hofstadler et al. (2003) “A highly efficient andautomated method of purifying and desalting PCR products for analysis byelectrospray ionization mass spectrometry” Anal Biochem. 316:50-57;Hofstadler et al. (2006) “Selective ion filtering by digitalthresholding: A method to unwind complex ESI-mass spectra and eliminatesignals from low molecular weight chemical noise” Anal Chem.78(2):372-378; and Hofstadler et al. (2005) “TIGER: The UniversalBiosensor” Int J Mass Spectrom. 242(1):23-41, which are eachincorporated by reference.

In addition to the molecular mass and base composition analyses referredto above, essentially any other nucleic acid amplification technologicalprocess is also optionally adapted for use in the systems of theinvention. Other exemplary uses of the systems and other aspects of theinvention include numerous biochemical assays, cell culture purificationsteps, and chemical synthesis, among many others. Many of these as wellas other exemplary applications of use in the systems of the inventionare also described in, e.g., Current Protocols in Molecular Biology,Volumes I, II, and III, 1997 (F. M. Ausubel ed.); Perbal, 1984, APractical Guide to Molecular Cloning; the series, Methods in Enzymology(Academic Press, Inc.); Sambrook et al., 2001, Molecular Cloning: ALaboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y.; Oligonucleotide Synthesis, 1984 (M. L. Gaited.); Nucleic Acid Hybridization, 1985, (Hames and Higgins);Transcription and Translation, 1984 (Hames and Higgins eds.); AnimalCell Culture, 1986 (R. I. Freshney ed.); Berger and Kimmel, Guide toMolecular Cloning Techniques, Methods in Enzymology volume 152 AcademicPress, Inc., San Diego, Calif. (Berger), DNA Cloning: A PracticalApproach, Volumes I and II, 1985 (D. N. Glover ed.); Immobilized Cellsand Enzymes, 1986 (IRL Press); Gene Transfer Vectors for MammalianCells, 1987 (J. H. Miller and M. P. Calos eds., Cold Spring HarborLaboratory); and Methods in Enzymology Vol. 154 and Vol. 155 (Wu andGrossman, and Wu, eds., respectively), which are each incorporated byreference.

Iii. Example Sample Processing Units and Carrier Mechanisms

FIGS. 1A-D schematically illustrate a sample processing unit orcomponents thereof according to one embodiment of the invention. Asshown, sample processing unit 100 includes cuvette 102 operablyconnected to first motor 104 (shown as a brushless DC motor). Firstmotor 104 (i.e., one embodiment of a rotational mechanism) is configuredto rotate cuvette 102 around a central longitudinal axis of cuvette 102.As also shown, first motor 104 is operably connected to support member106 (shown as a swing arm). First motor 104 is optionally configured(e.g., under the control of an appropriately programmed controller) torotate cuvette 102 in at least one pulsed mode, during which asubstantial portion of the time of rotation, a rate of rotation ofcuvette 102 exceeds a rate of rotation of sample material in cuvette 102such that the sample material is sheared away from a surface of cuvette102, e.g., to effect mixing of the sample material. To furtherillustrate, first motor 104 is optionally configured (e.g., again underthe control of an appropriately programmed controller) to rotate cuvette102 in at least one oscillating motion, e.g., also to effect mixing ofsample materials. Controllers and rotational modes are described furtherherein.

Support member 106 is also operably connected to second motor 108 (shownas a brushless direct current motor) via mounting bracket 110. Supportmember 106 includes first end 112 and second end 114. Cuvette 102 isretained proximal to first end 112 of support member 106 via first motor104, whereas second motor 108 is operably connected to support member106 proximal to second end 114 of support member 106. Support member 106is configured to rotate at least partially around a rotational axisextending through and proximal to second end 114 of support member 106.Pin 116 is fixedly coupled to second end 114 of support member 106 andaligned with the rotational axis. Pin 116 is also operably coupled tosecond motor 108, which effects rotation of cuvette 102 between secondposition 118 (e.g., a cuvette rotational or spin position) and firstposition 120 via pin 116 and support member 106. Collectively, secondmotor 108, pin 116, and support member 106 are components of oneembodiment of an exemplary conveyance mechanism. As additionally shown,sample processing unit 100 includes circuitry 122 that electricallyconnects to first motor 104, second motor 108, and a controller or powersource (not shown) to effect control of first motor 104 and second motor108.

Sample processing unit 100 also includes magnets 124 (shown as permanentmagnets) attached to magnet mounting arm 126, e.g., to facilitatecertain processing steps that involve magnetically-based separation ofmaterials. In some embodiments, electromagnets are utilized. Magnetmounting arm 126 is operably connected to mounting bracket 110 and holdsmagnets 124 in substantially fixed positions relative to cuvette 102 andsupport member 106. Magnets 124 are disposed proximal to receiving space126. As illustrated, for example, in FIG. 1 B and C, when cuvette 102 isin first position 120, cuvette 102 is located at least partially withinreceiving space 128.

In certain embodiments, the sample processing unit includes only asingle magnet attached to mounting bracket via a magnet mounting arm ina substantially fixed positions relative to a cuvette, support member,and first motor. In addition to a support member, the conveyancemechanism of an exemplary sample processing unit may also include asecond motor, which conveys a cuvette between a first position and asecond position.

The conveyance mechanisms of the sample processing units of theinvention include various embodiments. As mentioned above, in certainembodiments, conveyance mechanisms are configured to rotate cuvettes orother types of containers between selected positions (e.g., between spinmixing, detection, and magnetic particle retention positions).Essentially any other mechanism that can convey containers to and frombeing within magnetic communication with the magnets of the sampleprocessing units described herein is optionally utilized. As a furtherillustration, conveyance mechanisms include slidable support members insome embodiments. As shown in FIGS. 3A and B, for example, theconveyance mechanism of sample processing unit 300 includes supportmember 302 and gantry or linear slide track 304. Linear drive mechanism306 is configured to move support member 302 along linear slide track304. Further, rotational mechanism 308 (e.g., a motor or the like) isoperably coupled with container 310 via support member 302. In addition,magnet 312 is operably connected to linear slide track 304 in asubstantially fixed position via magnet mounting arm 314. Support member302 and container 310 are shown in first position 316 in FIG. 3A,whereas they are shown in second position 318 in FIG. 3B.

In certain embodiments, carrier mechanisms are operably connected tosample processing units. Carrier mechanisms are typically configured tomove sample processing units to one or more locations, e.g., wherevarious processing steps are performed, such as adding and/or removingfluidic materials from sample processing unit containers. Typically,multiple sample processing units are included on a given carriermechanism, e.g., to enhance the throughput of sample processingapplications performed using the carrier mechanism. In some embodiments,for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more sampleprocessing units are included on a given carrier mechanism. In addition,essentially any carrier mechanism format that can be used to move sampleprocessing units to selected locations is optionally utilized. In someembodiments, for example, a carrier mechanism includes a carousel thatis configured to rotate sample processing units to selected locations.In another representative embodiment, a carrier mechanism includes aconveyor track that is configured to convey sample processing units toone or more locations as desired. Both of these exemplary carriermechanism embodiments are described further herein. To furtherillustrate, manifolds for substantially simultaneously distributingfluidic materials to and/or from the containers of multiple sampleprocessing units of a given carrier mechanism are included in certainembodiments, e.g., to enhance process throughput.

One embodiment of a carrier mechanism with a manifold is schematicallydepicted in FIG. 4 A-F from various points of view. As shown, carousel400 includes 22 sample processing units 100 mounted on circular supportstructure 402, which is operably connected to rotating assembly 404.Rotating assembly 404 includes a slip ring or rotary electricalinterface that effects rotation of circular support structure 402 andsample processing units 100 to selected positions around carousel 400.Rotating assembly 404 is mounted on support base 406, which providesstructural support to carousel 400. Carousel 400 control includes motor408 (e.g., a stepper motor, such as a Model No. 5704M-10 or 5709L-06PDavailable from Lin Engineering, Santa Clara, Calif., U.S.A.) and one ormore transmissive photomicrosensors (e.g., a Model No. EE-SX1071available from Omron Electronics LLC, Schaumburg, Ill., U.S.A.).

As additionally shown, sample cleanup station or manifold 410 is alsomounted above carousel 400 on manifold support structure 412, which isconnected to rotating assembly 404 and manifold support pillars 414.Manifold 410 is used to aspirate and dispense fluidic materialsfrom/into cuvettes 102 of sample processing units 100 as part of samplepurification procedures. More specifically, manifold 410 includesaspirate heads 416 and dispense heads 418. Aspirate heads 416 typicallyfluidly communicate with fluidic material waste containers (not withinview) via flexible tubing, whereas dispense heads 418 generally fluidlycommunicate with fluidic material sources or reservoirs via flexibletubing. Fluidic material is typically conveyed through the tubing usinga fluid conveyance mechanism, such as a pump (e.g., a peristaltic pump,a vacuum pump, or the like). Manifold motor and linear motion component420, which includes manifold stepper motor (e.g., a Model No. 211-13-02or 211-20-02 available from Lin Engineering, Santa Clara, Calif.,U.S.A.), is configured to raise and lower manifold plate 424. As shown,aspirate heads 416 are mounted on manifold plate 424. When fluidicmaterials are aspirated from cuvettes 102, rotating assembly 404typically rotates selected cuvettes 102 into alignment with selectedaspirate heads 416. Manifold motor and linear motion component 420 thentypically lowers aspirate heads 416 such that needles of aspirate heads416 contact fluidic materials disposed within the selected cuvettes 102so that selected volumes of the fluidic materials can be aspirated fromthe selected cuvettes 102. In some of these embodiments, magneticallyresponsive particles (with bound or otherwise captured nucleic acids orother analytes) are included in the fluidic materials. In theseembodiments, the selected cuvettes 102 are typically moved into magneticcommunication with magnets 124 of the corresponding sample processingunits 100 so that the magnetically responsive particles are retainedwithin the selected cuvettes 102 as the selected aliquots are removedthrough the needles of the selected aspirate heads 416. After a givenfluidic material aspiration step is performed, manifold motor and linearmotion component 420 typically raises manifold plate 424 and aspirateheads 416 a sufficient distance such that rotating assembly 404 canrotate cuvettes 102 to other locations without contacting the needles ofaspirate heads 416. As further illustrated, dispense heads 418 aremounted in substantially fix positions on manifold support structure 412such that they can fluidly communicate with cuvettes 102 when cuvettes102 are positioned beneath and aligned with the needles of dispenseheads 418. During operation, e.g., before or after a given aspirationstep is performed, rotating assembly 404 typically rotates selectedcuvettes 102 into alignment with selected dispense heads 418 so thatselected volumes of fluidic material (e.g., reagent mixtures, elutionbuffers, etc.) can be dispensed into the selected cuvettes 102. Beforeor after a given aspiration or dispensing step is performed, selectedcuvettes 102 are typically spun using first motors 104 of sampleprocessing units 100 to mix fluidic materials in the selected cuvettes102. Exemplary systems that include carousels and manifolds aredescribed further herein.

To illustrate another exemplary embodiment, FIGS. 5 A and Bschematically depict a carrier mechanism that includes a conveyor trackfrom top and side elevation views, respectively. As shown, carriermechanism 500 includes conveyor track 502 (e.g., a conveyor belt, etc.),which is configured to rotate counter-clockwise around rotationalcouplings 504 (e.g., pulleys or the like). Sample processing units 506,which include containers 508, are connected to conveyor track 502.During operation, rotational couplings 504 rotate sample processingunits 506 to fluid transfer stations 510, 512, and 516, which includefluid transfer heads 518, 520, and 522, respectively, that each have anaspirate/dispense needle. Fluid transfer heads 518, 520, and 522 areconfigured to be raised and lowered. As shown, for example, in FIG. 5B,when sample processing units 506 are aligned beneath fluid transferheads 518, 520, and 522, the heads are typically lowered so that theaspirate/dispense needles can fluidly communicate with containers 508.Note that the container of the sample processing unit depicted on thenear side of conveyor track 502 in FIG. 5B partially obscures the needleof fluid transfer head 520, which is lowered into the container of thesample processing unit (not within view in FIG. 5B) on the far side ofconveyor track 502. When sample processing units 506 are rotated aroundrotational couplings 504, transfer heads 518, 520, and 522 are typicallyraised a sufficient height to permit the unobstructed passage ofcontainers 508 beneath the needles of transfer heads 518, 520, and 522.As also shown, transfer heads 518 and 522 are also configured to movealong gantry tracks 524 and 526, respectively.

IV. Example Controllers and Related Systems

Controllers are typically operably connected to sample processing unitsand carrier mechanisms, whether they are used as stand-alone sampleprocessing stations or as system components. The controllers of thesample processing stations and systems described herein are generallyconfigured to effect, e.g. the rotation of sample processing unitcontainers to mix sample materials in the containers (e.g., in variousselectable modes of rotation, etc.), the movement of containers to andfrom being within magnetic communication with the magnets of sampleprocessing units, the movement of carrier mechanisms to position sampleprocessing units relative to material transfer components, the transferof materials to and from the containers of sample processing units, thedetection of one or more parameters of sample materials disposed in thecontainers of sample processing units or of aliquots of those materialstaken from those containers, and the like. Controllers are typicallyoperably connected to one or more system components, such as motors(e.g., via motor drives), thermal modulating components, detectors,motion sensors, fluidic handling components, robotic translocationdevices, or the like, to control operation of these components. Morespecifically, controllers are generally included either as separate orintegral system components that are utilized to effect, e.g., therotation of the containers of sample processing units according to oneor more selectable rotational modes, the transfer of materials to and/orfrom the containers of sample processing units, the detection and/oranalysis of detectable signals received from sample materials bydetectors, etc. Controllers and/or other system components is/aregenerally coupled to an appropriately programmed processor, computer,digital device, or other logic device or information appliance (e.g.,including an analog to digital or digital to analog converter asneeded), which functions to instruct the operation of these instrumentsin accordance with preprogrammed or user input instructions (e.g.,mixing mode selection, fluid volumes to be conveyed, etc.), receive dataand information from these instruments, and interpret, manipulate andreport this information to the user.

A controller or computer optionally includes a monitor which is often acathode ray tube (“CRT”) display, a flat panel display (e.g., activematrix liquid crystal display, liquid crystal display, etc.), or others.Computer circuitry is often placed in a box, which includes numerousintegrated circuit chips, such as a microprocessor, memory, interfacecircuits, and others. The box also optionally includes a hard diskdrive, a floppy disk drive, a high capacity removable drive such as awriteable CD-ROM, and other common peripheral elements. Inputtingdevices such as a keyboard or mouse optionally provide for input from auser. An exemplary system comprising a computer is schematicallyillustrated in FIG. 6.

The computer typically includes appropriate software for receiving userinstructions, either in the form of user input into a set of parameterfields, e.g., in a GUI, or in the form of preprogrammed instructions,e.g., preprogrammed for a variety of different specific operations. Thesoftware then converts these instructions to appropriate language forinstructing the operation of one or more controllers to carry out thedesired operation, e.g., rotating sample processing unit containers tomix sample materials in the containers, aspirating fluidic materialsfrom sample processing unit containers, dispensing materials into sampleprocessing unit containers, or the like. The computer then receives thedata from, e.g., sensors/detectors included within the system, andinterprets the data, either provides it in a user understood format, oruses that data to initiate further controller instructions, inaccordance with the programming, e.g., such as in monitoring detectablesignal intensity, rates or modes of sample processing unit containerrotation, or the like.

More specifically, the software utilized to control the operation of thesample processing stations and systems of the invention typicallyincludes logic instructions that selectively direct, e.g., motors torotate cuvettes in pulsed modes, during which a substantial portion ofthe time of rotation, a rate of rotation of the cuvettes exceeds a rateof rotation of the samples in the cuvettes such that the samples aresheared away from surfaces of the cuvettes to effect sample mixing,motors to rotate the cuvettes in oscillating motions, and the like. Thelogic instructions of the software are typically embodied on a computerreadable medium, such as a CD-ROM, a floppy disk, a tape, a flash memorydevice or component, a system memory device or component, a hard drive,a data signal embodied in a carrier wave, and/or the like. Othercomputer readable media are known to persons of skill in the art. Insome embodiments, the logic instructions are embodied in read-onlymemory (ROM) in a computer chip present in one or more systemcomponents, without the use of personal computers.

The computer can be, e.g., a PC (Intel x86 or Pentium chip-compatibleDOS™, OS2™, WINDOWST™, WINDOWS NT™, WINDOWS98™, WINDOWS2000™, WINDOWSXP™, WINDOWS Vista™, LINUX-based machine, a MACINTOSHT™, Power PC, or aUNIX-based (e.g., SUN™ work station) machine) or other commoncommercially available computer which is known to one of skill. Standarddesktop applications such as word processing software (e.g., MicrosoftWord™ or Corel WordPerfect™) and database software (e.g., spreadsheetsoftware such as Microsoft Exce1™, Corel Quattro Pro™, or databaseprograms such as Microsoft Access™ or Paradox™) can be adapted to thepresent invention. Software for performing, e.g., sample processing unitcontainer rotation, material conveyance to and/or from sample processingunit containers, mixing process monitoring, assay detection, and datadeconvolution is optionally constructed by one of skill using a standardprogramming language such as Visual basic, C, C++, Fortran, Basic, Java,or the like.

The sample processing stations and related systems of the inventionoptionally include detectors or detection components configured todetect one or more detectable signals or parameters from a givenprocess, e.g., from materials disposed within sample processing unitcontainer or taken therefrom. In some embodiments, systems areconfigured to detect detectable signals or parameters that are upstreamand/or downstream of a given process involving the sample processingunits described herein. Suitable signal detectors that are optionallyutilized in these systems detect, e.g., pH, temperature, pressure,density, salinity, conductivity, fluid level, radioactivity,luminescence, fluorescence, phosphorescence, molecular mass, emission,transmission, absorbance, and/or the like. In some embodiments, thedetector monitors a plurality of signals, which correspond in positionto “real time” results. Example detectors or sensors include PMTs, CCDs,intensified CCDs, photodiodes, avalanche photodiodes, optical sensors,scanning detectors, or the like. Each of these as well as other types ofsensors is optionally readily incorporated into the sample processingstations and systems described herein. The detector optionally movesrelative to the stations, sample containers or other assay components,or alternatively, the stations, sample containers or other assaycomponents move relative to the detector. Optionally, the stations andsystems of the invention include multiple detectors. In these stationsand systems, such detectors are typically placed either in or adjacentto, e.g., a sample processing unit cuvette or other vessel, such thatthe detector is in sensory communication with the sample processing unitcuvette or other vessel (i.e., the detector is capable of detecting theproperty of the cuvette or vessel or portion thereof, the contents of aportion of the cuvette or vessel, or the like, for which that detectoris intended).

The detector optionally includes or is operably linked to a computer,e.g., which has system software for converting detector signalinformation into assay result information or the like. For example,detectors optionally exist as separate units, or are integrated withcontrollers into a single instrument. Integration of these functionsinto a single unit facilitates connection of these instruments with thecomputer, by permitting the use of a few or even a single communicationport for transmitting information between system components. Detectioncomponents that are optionally included in the systems of the inventionare described further in, e.g., Skoog et al., Principles of InstrumentalAnalysis, 6^(th) Ed., Brooks Cole (2006) and Curren, AnalyticalInstrumentation:

Performance Characteristics and Quality, John Wiley & Sons, Inc. (2000),which are both incorporated by reference.

The sample processing stations and systems of the invention optionallyalso include at least one robotic translocation or gripping componentthat is structured to grip and translocate containers or otherprocessing components between components of the stations or systemsand/or between the stations or systems and other locations (e.g., otherwork stations, etc.). A variety of available robotic elements (roboticarms, movable platforms, etc.) can be used or modified for use withthese systems, which robotic elements are typically operably connectedto controllers that control their movement and other functions.

FIG. 6 is a schematic showing a representative system including aninformation appliance in which various aspects of the present inventionmay be embodied. Other exemplary systems are also described herein. Aswill be understood by practitioners in the art from the teachingsprovided herein, the invention is optionally implemented in hardware andsoftware. In some embodiments, different aspects of the invention areimplemented in either client-side logic or server-side logic. As willalso be understood in the art, the invention or components thereof maybe embodied in a media program component (e.g., a fixed media component)containing logic instructions and/or data that, when loaded into anappropriately configured computing device, cause that apparatus orsystem to perform according to the invention. As will additionally beunderstood in the art, a fixed media containing logic instructions maybe delivered to a viewer on a fixed media for physically loading into aviewer's computer or a fixed media containing logic instructions mayreside on a remote server that a viewer accesses through a communicationmedium in order to download a program component.

FIG. 6 shows information appliance or digital device 600 that may beunderstood as a logical apparatus (e.g., a computer, etc.) that can readinstructions from media 617 and/or network port 619, which canoptionally be connected to server 620 having fixed media 622.Information appliance 600 can thereafter use those instructions todirect server or client logic, as understood in the art, to embodyaspects of the invention. One type of logical apparatus that may embodythe invention is a computer system as illustrated in 600, containing CPU607, optional input devices 609 and 611, disk drives 615 and optionalmonitor 605. Fixed media 617, or fixed media 622 over port 619, may beused to program such a system and may represent a disk-type optical ormagnetic media, magnetic tape, solid state dynamic or static memory, orthe like. In specific embodiments, the aspects of the invention may beembodied in whole or in part as software recorded on this fixed media.Communication port 619 may also be used to initially receiveinstructions that are used to program such a system and may representany type of communication connection. Optionally, aspects of theinvention are embodied in whole or in part within the circuitry of anapplication specific integrated circuit (ACIS) or a programmable logicdevice (PLD). In such a case, aspects of the invention may be embodiedin a computer understandable descriptor language, which may be used tocreate an ASIC, or PLID.

In addition, FIG. 6 also shows sample processing station 602, which isoperably connected to information appliance 600 via server 620.Optionally, sample processing station 602 is directly connected toinformation appliance 600. During operation, sample processing station602 typically mixes and retains selected materials (e.g., magneticallyresponsive particles with captured target materials, etc.) in thecuvettes of the sample processing units of sample processing station602, e.g., as part of an assay or other process. FIG. 6 also showsmaterial transfer component 623 and detector 624, which are optionallyincluded in the systems of the invention. As shown, material transfercomponent 623 and detector 624 are operably connected to informationappliance 600 via server 620. In some embodiments, material transfercomponent 623 and/or detector 624 is directly connected to informationappliance 600. Material transfer component 623 is typically configuredto transfer materials to and/or from the cuvettes of the sampleprocessing units of sample processing station 602. In certainembodiments, detector 624 is configured to detect detectable signalsproduced in the cuvettes of the sample processing units of sampleprocessing station 602 or in aliquots of materials removed from and/orto be added to those cuvettes.

V. Example Sample Processing System and Related Process Embodiments

To further illustrate exemplary embodiments of the invention, FIG. 7 A-Gschematically depict a portion of a representative system for nucleicacid amplification product desalting and molecular mass measurement thatincludes a sample processing station as a sub-system component. Themeasured molecular masses of the amplification products are typicallyused to determine base compositions of the corresponding amplificationproducts, which are then generally correlated with the identities ororganismal sources of the initial template nucleic acids, for example,as part of a research or in-vitro diagnostic application, among manyothers.

As shown in FIG. 7 A-G, components of representative system 700 includemicroplate handling component or system 10, material transfer component702, mixing station 704, wash stations 706 and 708, sample processingcomponent 710, and sample injector 712. During operation, microplatesare typically stored or positioned in input non-priority microplatestorage unit 12, output non-priority microplate storage unit 14,priority microplate storage unit 16, microplate processing area 18, andnon-priority microplate holding area 20 (e.g., on non-prioritymicroplate holding component 22) of microplate handling component 10. Asalso shown, microplate handling component 10 also includes barcodereader 36. In the exemplary embodiment shown, barcode reader 36 isconfigured to read barcodes disposed on microplates when the microplatesare disposed in or proximal to non-priority microplate holding area 20,e.g., to track the microplates or samples contained in the microplatesin microplate handling system 10. In some embodiments, for example,non-priority microplates are stored in input non-priority microplatestorage unit 12 and priority microplates are stored in prioritymicroplate storage unit 16 after target regions of template nucleicacids in those plates have been amplified, e.g., at a separatethermocycling station or nucleic acid amplification component.Essentially any thermal cycling station or device is optionally adaptedfor use with a system of the invention, such as system 700. Examples ofsuitable thermocycling devices that are optionally utilized areavailable from many different commercial suppliers, includingMastercycler® devices (Eppendorf North America, Westbury, N.Y., U.S.A.),the COBAS® AMPLICOR Analyzer (Roche Molecular Systems, Inc., Pleasanton,Calif., U.S.A.), Mycycler and iCycler Thermal Cyclers (Bio-RadLaboratories, Inc., Hercules, Calif., U.S.A.), and the SmartCyclerSystem (Cepheid, Sunnyvale, Calif., U.S.A.), among many others. In otherexemplary embodiments, sample preparation components, nucleic acidamplification components, and related fluid handling or materialtransfer components are integrated with the systems described herein,e.g., to fully automate a given nucleic acid amplification and analysisprocess. Instruments that can be adapted for this purpose include, forexample, the m2000™ automated instrument system (Abbott Laboratories,Abbott Park, Ill., U.S.A.), the GeneXpert System (Cepheid, Sunnyvale,Calif. U.S.A.), and the COBAS® AmpliPrep® System (Roche MolecularSystems, Inc., Pleasanton, Calif., U.S.A.), and the like.

Microplates are transferred from input non-priority microplate storageunit 12 or priority microplate storage unit 16 to microplate processingarea 18 using platform 28 of a microplate transport mechanism. Asreferred to above and as shown in, e.g., FIGS. 7 F and G, platform 28 isoperably connected to X-axis linear motion component 38. X-axis linearmotion component 38 includes gantry 40. Platform 28 is operablyconnected to carriage 42, which moves along gantry 40, driven by motor89. As further shown in FIGS. 7 F and G, microplate transport mechanism26 also includes Y-axis linear motion component 44 operably connected tocarriage 42 and to platform 28. Y-axis linear motion component 44 isconfigured to raise and lower platform 28 along the Y-axis. Suitablelinear motion components, motors, and motor drives are generallyavailable from many different commercial suppliers including, e.g.,Techno-Isel Linear Motion Systems (New Hyde Park, N.Y., U.S.A.), NCServo Technology Corp. (Westland, Mich., USA), Enprotech AutomationServices (Ann Arbor, Mich., U.S.A.), Yaskawa Electric America, Inc.(Waukegan, Ill., U.S.A.), ISL Products International, Ltd. (Syosset,N.Y., U.S.A.), AMK Drives & Controls, Inc. (Richmond, Va., U.S.A.),Aerotech, Inc. (Pittsburgh, Pa., U.S.A.), HD Systems Inc. (Hauppauge,N.Y., U.S.A.), and the like. Additional detail relating to motors andmotor drives are described in, e.g., Polka, Motors and Drives, ISA(2002) and Hendershot et al., Design of Brushless Permanent-MagnetMotors, Magna Physics Publishing (1994), which are both incorporated byreference. Microplate handling components are also described in, e.g.,U.S. Provisional Patent Application No. 61/097,523, entitled “MICROPLATEHANDLING SYSTEMS AND RELATED COMPUTER PROGRAM PRODUCTS AND METHODS”filed Sep. 16, 2008 by Hofstadler et al., which is incorporated byreference in its entirety. FIG. 7G also shows power source 98, whichprovides power for the system shown in this figure.

Material transfer component 702 includes sample input gantry 714 andsample output gantry 716. Input gantry head 718 is configured to movealong sample input gantry 714, whereas output gantry head 720 isconfigured to move along sample output gantry 716. Input gantry head 718and output gantry head 720 each include needles that are configured toaspirate and dispense fluidic materials. Further, input gantry head 718and output gantry head 720 are each configured to be raised and loweredalong the Y-axis. During operation of exemplary system 700, the needleor pipetting tip of input gantry head 718 is typically used to aspiratean aliquot of magnetically responsive particles (e.g., magneticallyresponsive beads, such as BioMag®Plus Amine superparamagneticmicroparticles available from Bangs Laboratories, Inc., Fishers, Ind.,U.S.A.) that bind nucleic acids from a mixing cartridge positioned atmixing station 704. Magnetically responsive particle sources and mixingstations are also described in, e.g., U.S. Provisional PatentApplication No. 61/097,520, entitled “MIXING CARTRIDGES, MIXINGSTATIONS, AND RELATED KITS, SYSTEMS, AND METHODS” filed Sep. 16, 2008 byHofstadler et al., which is incorporated by reference in its entirety.Nucleic acid purification involving magnetically responsive particles isalso described in, e.g., U.S. Patent App. Pub. No. US 2005/0164215,entitled “METHOD FOR RAPID PURIFICATION OF NUCLEIC ACIDS FOR SUBSEQUENTANALYSIS BY MASS SPECTROMETRY BY SOLUTION CAPTURE,” filed May 12, 2004by Hofstadler et al., and U.S. Patent App. Pub. No. US 2005/0130196,entitled “METHOD FOR RAPID PURIFICATION OF NUCLEIC ACIDS FOR SUBSEQUENTANALYSIS BY MASS SPECTROMETRY BY SOLUTION CAPTURE,” filed Sep. 17, 2004by Hofstadler et al., which are both incorporated by reference in theirentirety. Optionally before, but typically after aspirating the aliquotof magnetically responsive particles (e.g., to minimize the possibilityof cross-contaminating samples), the needle of input gantry head 718 isalso generally used to aspirate an aliquot of an amplification productsample from a selected well of a microplate positioned in microplateprocessing area 18 of microplate handling system 10. The resultingmixture of magnetically responsive particle and amplification productsample aliquots disposed within the needle of input gantry head 718 isthen typically transferred to sample processing component 710 alongsample input gantry 714. After dispensing the mixture at sampleprocessing component 710, the needle of input gantry head 718 istypically washed at wash station 706, e.g., to minimize the probabilityof cross-contaminating samples, prior to repeating this transfer cyclefor other amplification product samples contained in the wells of agiven microplate (e.g., priority or non-priority microplates) positionedin microplate processing area 18 of microplate handling system 10.

In the embodiment shown, sample processing station or component 710 is adesalting station that is used to desalt or otherwise purify nucleicacid amplification products in the sample mixture prior to massspectrometric analysis. Sample processing component 710 includes carriermechanism 722 (shown as a carousel), which includes a plurality ofsample processing units 724. In the illustrated embodiment, each sampleprocessing unit 724 includes cuvette 726 and magnet 728. After a mixtureof magnetically responsive particle and amplification product samplealiquots is dispensed into a given cuvette 726, that cuvette istypically rotated in a counter-clockwise direction on carrier mechanism722 to various positions within sample processing component 710 wherevarious reagents (e.g., washes with ammonium bicarbonate and/or MeOH,etc.) are added to and/or removed from that cuvette (e.g., via variousfluidic handling components of manifold 730) as part of the process ofpurifying the amplification products captured or otherwise bound to themagnetically responsive particles in the mixture. When fluidic materialsare removed from the cuvette at a given position within sampleprocessing component 710, the cuvette is typically moved proximal to themagnet of the particular sample processing unit (e.g., cuvette 726 ismoved proximal to magnet 728 of sample processing unit 724) using aconveyance mechanism to establish sufficient magnetic communicationbetween the magnet and the magnetically responsive particles such thatthe magnetically responsive particles are moved to and retained on aninternal surface of the cuvette while fluidic materials are removed fromthe cuvette. At the conclusion of a purification process for a givensample, the purified amplification products are then typically aspiratedfrom the particular cuvette using the needle of output gantry head 720.During or prior this step, the nucleic acid amplification products areeluted (e.g., using a solution that includes piperidine, imidazole,MeOH, and optionally peptide calibration standards (used as part ofsubsequent mass spectrometric analyses), or the like) from themagnetically responsive particles. After purified amplification productshave been removed from a given cuvette, that cuvette is then generallyrotated on carrier mechanism 722 into communication with cuvette washstation 727, where the cuvette is washed prior to commencing anotherpurification cycle involving the cuvette and another sample. Sampledesalting/purification methods are also described in, e.g., U.S. PatentApp. Pub. No. US 2005/0164215, entitled “METHOD FOR RAPID PURIFICATIONOF NUCLEIC ACIDS FOR SUBSEQUENT ANALYSIS BY MASS SPECTROMETRY BYSOLUTION CAPTURE,” filed May 12, 2004 by Hofstadler et al., and U.S.Patent App. Pub. No. US 2005/0130196, entitled “METHOD FOR RAPIDPURIFICATION OF NUCLEIC ACIDS FOR SUBSEQUENT ANALYSIS BY MASSSPECTROMETRY BY SOLUTION CAPTURE,” filed Sep. 17, 2004 by Hofstadler etal., and Hofstadler et al. (2003) “A highly efficient and automatedmethod of purifying and desalting PCR products for analysis byelectrospray ionization mass spectrometry” Anal Biochem. 316:50-57,which are each incorporated by reference in their entirety.

Purified and eluted amplification products that have been aspirated froma particular cuvette of sample processing component 710 are typicallytransported along sample output gantry 716 to sample injector 712 (shownas a two channel time-of-flight injector) using output gantry head 720.That is, the amplification products are typically dispensed from theneedle or pipetting tip of output gantry head 720 into one of the twochannels of sample injector 712, which generally comprise twoindependent sample injection syringe pumps that are configured toreceive the amplification products. After dispensing the amplificationproducts at sample injector 712, the needle of output gantry head 720 istypically washed at wash station 708 prior to aspirating anotherpurified amplification product sample from sample processing component710, e.g., to reduce the potential for carryover contamination betweensamples.

Now referring to FIG. 8, which schematically shows additional componentsof representative system 700 (sample processing component 710 is notshown so that other system components are within view) from aperspective view. As shown, the additional components include dualsprayer module 732, which includes two independent electrosprayionization sprayers, and time-of-flight mass spectrometer 734.Amplification product samples received at sample injector 712 aretypically injected into one of the two sprayers of dual sprayer module732 for electrospray ionization and mass measurement in time-of-flightmass spectrometer 734. As further shown, the additional components ofrepresentative system 700 also include input/output device 736 (shown asa touch screen monitor), computer 737, output device 739 (shown as aprinter), reagent and/or waste modules 738, and chassis 740.Input/output device 736, computer 737, and output device 739 arecomponents of a controller of system 700. Controllers are describedfurther herein. Reagent module 738 provides reagent sources, and thewaste module 738 provides waste receptacles for system 700. Chassis 740provides mechanical support for microplate handling system 10, sampleprocessing component 710, and other components of system 700. To furtherillustrate, FIGS. 11A-C schematically show representative system 700with an external covering from various views.

In some embodiments, the base compositions of amplification products aredetermined from detected molecular masses. In these embodiments, basecompositions are typically correlated with the identity of an organismalsource, genotype, or other attribute of the corresponding templatenucleic acids in a given sample. Suitable software and related aspects,e.g., for determining base compositions from detected molecular massesand for performing other aspects of base composition analysis arecommercially available from Ibis Biosciences, Inc. (Carlsbad, Calif.,U.S.A.). Nucleic acid base composition analysis is also described inmany of the publications referred to herein, including, e.g., U.S. Pat.No. 7,255,992, entitled “METHODS FOR RAPID DETECTION AND IDENTIFICATIONOF BIOAGENTS FOR ENVIRONMENTAL AND PRODUCT TESTING,” which issued Aug.14, 2007 to Ecker et al., U.S. Pat. No. 7,226,739, entitled “METHODS FORRAPID DETECTION AND IDENTIFICATION OF BIOAGENTS IN EPIDEMIOLOGICAL ANDFORENSIC INVESTIGATIONS,” which issued Jun. 5, 2007 to Ecker et al.,U.S. Pat. No. 7,217,510, entitled “METHODS FOR PROVIDING BACTERIALBIOAGENT CHARACTERIZING INFORMATION,” which issued May 15, 2007 to Eckeret al., and U.S. Pat. No. 7,108,974, entitled “METHOD FOR RAPIDDETECTION AND IDENTIFICATION OF BIOAGENTS,” which issued Sep. 19, 2006to Ecker et al., which are each incorporated by reference in theirentirety.

VI. Fabrication Methods and Materials

Sample processing units or components thereof, carrier mechanisms orcomponents thereof, and station or system components (e.g., mixingstations, microplate storage units, microplate transport mechanisms,support bases, etc.) are optionally formed by various fabricationtechniques or combinations of such techniques including, e.g.,machining, embossing, extrusion, stamping, engraving, injection molding,cast molding, etching (e.g., electrochemical etching, etc.), or othertechniques. These and other suitable fabrication techniques aregenerally known in the art and described in, e.g., Molinari et al.(Eds.), Metal Cutting and High Speed Machining, Kluwer AcademicPublishers (2002), Altintas, Manufacturing Automation: Metal CuttingMechanics, Machine Tool Vibrations, and CNC Design, Cambridge UniversityPress (2000), Stephenson et al., Metal Cutting Theory and Practice,Marcel Dekker (1997), Fundamentals of Injection Molding, W.

J. T. Associates (2000), Whelan, Injection Molding of ThermoplasticsMaterials, Vol. 2, Chapman & Hall (1991), Rosato, Injection MoldingHandbook, 3^(rd) Ed., Kluwer Academic Publishers (2000), Fisher,Extrusion of Plastics, Halsted Press (1976), and Chung, Extrusion ofPolymers: Theory and Practice, Hanser-Gardner Publications (2000), whichare each incorporated by reference. Exemplary materials optionally usedto fabricate sample processing units, carrier mechanisms, manifolds, orcomponents thereof include metal (e.g., steel, aluminum, etc.), glass,polymethylmethacrylate, polyethylene, polydimethylsiloxane,polyetheretherketone, polytetrafluoroethylene, polystyrene,polyvinylchloride, polypropylene, polysulfone, polymethylpentene, andpolycarbonate, among many others. In certain embodiments, followingfabrication, system components are optionally further processed, e.g.,by coating surfaces with a hydrophilic coating, a hydrophobic coating(e.g., a Xylan 1010DF/870 Black coating available from WhitfordCorporation (West Chester, Pa.), etc.), or the like, e.g., to preventinteractions between component surfaces and reagents, samples, or thelike.

VII. Examples

It is understood that the examples and embodiments described herein arefor illustrative purposes only and are not intended to limit the scopeof the claimed invention. It is also understood that variousmodifications or changes in light the examples and embodiments describedherein will be suggested to persons skilled in the art and are to beincluded within the spirit and purview of this application and scope ofthe appended claims.

I. PCR Product Purification and Desalting Example 1. PCR ProductPurification

PCR products were thoroughly purified and desalted before ESI MS. Thisstep typically precedes ESI-MS analysis, because PCR salts and buffercomponents generally have a deleterious effect on the ESI process. Evensmall amounts of salts (<1 gmol/L) will typically significantly reduceESI sensitivity, owing to the appearance of multiple cation adducts inthe mass spectra. The protocol used in this example is based on a weakanion-exchange method, in which amplified DNA was bound to a weakanion-exchange resin coated on the outside of magnetic bead particles.Unconsumed deoxynucleoside triphosphates, salts, and otherlow-molecular-weight species that could interfere with subsequent ESI-MSanalysis were removed using a sample processing station system describedherein and the PCR cleanup process outlined as follows:

1. Loaded 40 μL PCR Product and 50 μL magnetic bead solution into aclean cuvette;2. Mixed the beads for 4.5 minutes to allow the DNA to bind the magneticbeads;3. Positioned the cuvette at the magnet for 30 seconds to separate thebeads from the solution;4. Aspirated the liquid from the cuvette and dispensed 80 μL of 100 mMammonium bicarbonate in a 50:50 methanol:water solution;5. Resuspended and washed the beads by mixing for 35 seconds;6. Positioned the cuvette at the magnet for 15 seconds to separate thebeads from the solution;7. Aspirated the liquid from the cuvette and dispensed 80 μL of 100 mMammonium bicarbonate in a 50:50 methanol:water solution;8. Resuspended and washed the beads by mixing for 35 seconds;9. Positioned the cuvette at the magnet for 15 seconds to separate thebeads from the solution;10. Aspirated the liquid from the cuvette and dispensed 80 μL of 50:50methanol:water solution;11. Resuspended and washed the beads by mixing for 35 seconds;12. Positioned the cuvette at the magnet for 15 seconds to separate thebeads from the solution;13. Aspirated the liquid from the cuvette and dispensed 40 μL of 25 mMpiperidine and 25 mM imidazole in 35:65 methanol:water solution;14. Resuspended the magnetic beads and allowed time for the DNA to elutefrom the beads for 2 minutes;15. Positioned the cuvette at the magnet for 30 seconds to separate thebeads from the solution; and16. Aspirated the solution and injected into the ESI-MS for analysis.TOTAL PCR PRODUCT CLEANUP TIME=10 minutes

II. 2. Cuvette Cleaning Protocol

The cuvette cleaning protocol utilized to clean the cuvette after PCRproducts were purified and desalted was as follows:

1. Dispensed 160 μL of 25 mM piperidine and 25 mM imidazole in 35:65methanol:water solution and mixed for 15 seconds;2. Aspirated solution and dispensed 160 μL of clean (Type I) water intothe cuvette;3. Mixed for 15 seconds; and4. Aspirated liquid from cuvette.TOTAL CUVETTE CLEANUP TIME=2 minutes

II. 3. ESI-TOF Mass Spectrometry

A Bruker Daltonics (Billerica, Mass., U.S.A.) MicroTOF-ESItime-of-flight (TOF) mass spectrometer was used to analyze purified anddesalted PCR products in this example. Ions from the ESI sourceunderwent orthogonal ion extraction and were focused in a reflectronprior to detection. Ions were formed in the standard MicroTOF-ESIsource, which was equipped with an off-axis sprayer and glass capillary.For operation in the negative ion mode, the atmospheric pressure end ofthe glass capillary was biased at 3500 V relative to the ESI needleduring data acquisition. A countercurrent flow of dry N₂ gas wasemployed to assist in the desolvation process. External ion accumulationwas employed to improve ionization duty cycle during data acquisitionand to enhance sensitivity in the m/z range of interest. In thisexample, each 75 μs scan was comprised of 75,000 data points (a 37.5 μsdelay followed by a 37.5 μs digitization event at 2 GHz). For eachspectrum, 660 000 scans were co-added. Example data obtained from thisanalysis is shown in FIG. 2.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the techniques and apparatus described abovecan be used in various combinations. All publications, patents, patentapplications, and/or other documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication, patent, patent application,and/or other document were individually indicated to be incorporated byreference for all purposes.

1-20. (canceled)
 21. A sample processing unit, comprising: at least onecontainer configured to contain at least one sample comprising at leastone magnetically responsive particle; at least one magnet thatgenerates, or is configured to generate, at least one magnetic field,which magnet is in a substantially fixed position relative to thecontainer; at least one conveyance mechanism configured to convey thecontainer between at least first and second positions, wherein at leastthe first position is within magnetic communication with the magnet whenthe magnet generates the magnetic field; and, at least one rotationalmechanism operably connected to the container, which rotationalmechanism is configured to rotate the container when the container is inat least the second position.